ARF prevents tumorigenesis through p53-dependent and -independent pathways. ARF's ability to activate p53 by negatively regulating MDM2 in response to oncogenes has been well studied. However, recent analysis of ARF's p53-independent functions has uncovered a seemingly disparate number of protein targets involved in numerous cellular processes. To overcome this limitation, we have sought to functionally link several of the p53-independent targets of ARF. We have shown that ARF limits the activity of the miRNA Drosha-DDX5 processing complex to prevent cellular transformation and we have established that ARF inhibits innate immune signaling in the face of increased double-stranded RNA sensing to suppress enhanced tumorigenesis. We hypothesize that ARF's p53-independent targets contain a commonality that can be used to inhibit tumorigenesis. We are ideally positioned to solve this problem, having extensive experience in both the ARF field and in applying this knowledge to the study of cancer biology. Our established track record involves the identification of Mdm2 nucleolar sequestration by ARF to activate p53, the discovery of the p53-independent function of ARF, and the targeting of NPM, DDX5, Drosha, and IFN-? as p53-independent targets of ARF. We will determine how seemingly unique ARF targets act to stimulate IFN-? production through a cytosolic miRNA or double-stranded RNA sensing mechanism. We will also establish whether IFN-? activation is a result of a more generalized increase in cytosolic RNAs and determine the mechanism for cytosolic RNA accumulation. The identification of specific miRNAs or accumulated cytosolic RNAs responsible for IFN-? production will be key in determining the mechanism of enhanced tumorigenesis in the absence of ARF and p53. We will identify proteins that are ISGylated and how they drive tumorigenesis. The discovery of ISG15-modified proteins in tumor formation is completely novel in this context and would indicate a prominent role for this pathway in tumor cell biology. We will establish which ARF targets serve as valid biomarkers of ARF/p53 loss of function in human breast cancer and whether any of these predict patient survival. A comprehensive analysis of ARF/p53, its targets, and IFN-? pathway components within the same human breast cancers will provide the research community with the first much-needed prognostic tool for this novel network of suppressors and drivers. Finally, we will use JAK1/2 inhibitors to inhibit the tumorigenesis of established and isogenic human cancer cell lines in vitro as well as spontaneous and xenografted in vivo tumors. These pre-clinical studies have the potential to test JAK1/2 inhibitors in a setting where we have shown a genetic dependence on the pathway. Additionally, our assays will also be able to identify complimentary pathways that might provide resistance to JAK1/2 inhibition.